Project Summary/Abstract Transcriptomic-based approaches, and in recent years, single-cell RNA sequencing, are revolutionizing our understanding of cellular heterogeneity, opening up a new route to identify novel cell markers at unprecedented scale across many different cell types. ?RNA-based live-cell sorting opens up >99% of the marker space to enable higher specificity cell sorting. However, existing methods for cell-based RNA detection are either incompatible with live cells due to fixation and permeabilization (RNA-FISH) or suffer from poor signal-to-noise (S/N) and specificity (molecular beacons, SmartFlares). Researchers are currently limited to purifying cells using antibody-based detection of cell surface protein markers via fluorescence activated cell sorting (FACS) and magnetic activated cell sorting (MACS). These cell-surface protein markers are often not always specific enough to isolate important cell subsets as they are also often expressed on non-target cell types. We propose to develop a robust and easy-to-use live-cell reagent kit leveraging the specificity of CRISPR-Cas9 to detect RNA in individual cells for FACS-based isolation. While Cas9 is best known as a programmable sequence-specific DNA endonuclease for gene editing applications, Cas9 can be re-directed to bind and cut RNA by hybridization of a protospacer-adjacent motif (PAM; a sequence required for Cas9 DNA cleavage)-containing DNA oligonucleotide (a ?PAMmer?) to the target RNA (RCas9). By modifying the PAMmer with a quencher and fluorophore (FQ-PAMmer), we aim to use Cas9?s cleavage activity to release a quencher (Q) and activate a fluorescent (F) signal in live cells only upon specific Cas9 guide RNA-mediated binding of target RNA. While our technology platform is broadly applicable to theoretically any RNA target, our proof-of-principle studies will be focused on the isolation of a specific T cell subpopulation expressing ?IFNG mRNA. The objective of this Phase I STTR project is to demonstrate isolation of live ?IFNG mRNA+ T cells with FACS from heterogeneous T cell cultures. The project is organized in two aims to first identify candidate RCas9 FQ-PAMmer probes targeting the length of the IFNG mRNA transcript with high S/N ?in vitro and stability in live cells (Aim 1), then test the RCas9 FQ-PAMmer reagents in live cells and demonstrate isolation of live ?IFGN mRNA+ T cells via FACS (Aim 2). ?Commercialization of Dahlia Biosciences? live-cell detection reagent kits will provide a critical tool to research and drug development scientists to isolate and characterize functionally important rare cell populations, including T cells.